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• W3011916202 abstract "•Reconstitution of RNAPII pausing in vitro, with purified factors (no extracts)•Human PIC alone is sufficient to establish RNAPII pausing•TFIID is required for RNAPII promoter-proximal pausing•Rapid TFIID depletion induces RNAPII pause release genome-wide RNA polymerase II (RNAPII) transcription is governed by the pre-initiation complex (PIC), which contains TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH, RNAPII, and Mediator. After initiation, RNAPII enzymes pause after transcribing less than 100 bases; precisely how RNAPII pausing is enforced and regulated remains unclear. To address specific mechanistic questions, we reconstituted human RNAPII promoter-proximal pausing in vitro, entirely with purified factors (no extracts). As expected, NELF and DSIF increased pausing, and P-TEFb promoted pause release. Unexpectedly, the PIC alone was sufficient to reconstitute pausing, suggesting RNAPII pausing is an inherent PIC function. In agreement, pausing was lost upon replacement of the TFIID complex with TATA-binding protein (TBP), and PRO-seq experiments revealed widespread disruption of RNAPII pausing upon acute depletion (t = 60 min) of TFIID subunits in human or Drosophila cells. These results establish a TFIID requirement for RNAPII pausing and suggest pause regulatory factors may function directly or indirectly through TFIID. RNA polymerase II (RNAPII) transcription is governed by the pre-initiation complex (PIC), which contains TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH, RNAPII, and Mediator. After initiation, RNAPII enzymes pause after transcribing less than 100 bases; precisely how RNAPII pausing is enforced and regulated remains unclear. To address specific mechanistic questions, we reconstituted human RNAPII promoter-proximal pausing in vitro, entirely with purified factors (no extracts). As expected, NELF and DSIF increased pausing, and P-TEFb promoted pause release. Unexpectedly, the PIC alone was sufficient to reconstitute pausing, suggesting RNAPII pausing is an inherent PIC function. In agreement, pausing was lost upon replacement of the TFIID complex with TATA-binding protein (TBP), and PRO-seq experiments revealed widespread disruption of RNAPII pausing upon acute depletion (t = 60 min) of TFIID subunits in human or Drosophila cells. These results establish a TFIID requirement for RNAPII pausing and suggest pause regulatory factors may function directly or indirectly through TFIID. RNA polymerase II (RNAPII) transcribes all protein-coding and many non-coding RNAs in the human genome. RNAPII transcription initiation occurs within the pre-initiation complex (PIC), which contains TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH, RNAPII, and Mediator. After initiation, RNAPII enzymes typically pause after transcribing 20–80 bases (Kwak and Lis, 2013Kwak H. Lis J.T. Control of transcriptional elongation.Annu. Rev. Genet. 2013; 47: 483-508Crossref PubMed Scopus (272) Google Scholar), and paused polymerases represent a common regulatory intermediate (Core et al., 2008Core L.J. Waterfall J.J. Lis J.T. Nascent RNA sequencing reveals widespread pausing and divergent initiation at human promoters.Science. 2008; 322: 1845-1848Crossref PubMed Scopus (1415) Google Scholar, Jonkers et al., 2014Jonkers I. Kwak H. Lis J.T. Genome-wide dynamics of Pol II elongation and its interplay with promoter proximal pausing, chromatin, and exons.eLife. 2014; 3: e02407Crossref PubMed Scopus (342) Google Scholar, Muse et al., 2007Muse G.W. Gilchrist D.A. Nechaev S. Shah R. Parker J.S. Grissom S.F. Zeitlinger J. Adelman K. RNA polymerase is poised for activation across the genome.Nat. Genet. 2007; 39: 1507-1511Crossref PubMed Scopus (569) Google Scholar, Zeitlinger et al., 2007Zeitlinger J. Stark A. Kellis M. Hong J.W. Nechaev S. Adelman K. Levine M. Young R.A. RNA polymerase stalling at developmental control genes in the Drosophila melanogaster embryo.Nat. Genet. 2007; 39: 1512-1516Crossref PubMed Scopus (574) Google Scholar). Accordingly, paused RNAPII has been implicated in enhancer function (Ghavi-Helm et al., 2014Ghavi-Helm Y. Klein F.A. Pakozdi T. Ciglar L. Noordermeer D. Huber W. Furlong E.E. Enhancer loops appear stable during development and are associated with paused polymerase.Nature. 2014; 512: 96-100Crossref PubMed Scopus (334) Google Scholar, Henriques et al., 2018Henriques T. Scruggs B.S. Inouye M.O. Muse G.W. Williams L.H. Burkholder A.B. Lavender C.A. Fargo D.C. Adelman K. Widespread transcriptional pausing and elongation control at enhancers.Genes Dev. 2018; 32: 26-41Crossref PubMed Scopus (180) Google Scholar), development and homeostasis (Adelman et al., 2009Adelman K. Kennedy M.A. Nechaev S. Gilchrist D.A. Muse G.W. Chinenov Y. Rogatsky I. Immediate mediators of the inflammatory response are poised for gene activation through RNA polymerase II stalling.Proc. Natl. Acad. Sci. USA. 2009; 106: 18207-18212Crossref PubMed Scopus (115) Google Scholar, Lagha et al., 2013Lagha M. Bothma J.P. Esposito E. Ng S. Stefanik L. Tsui C. Johnston J. Chen K. Gilmour D.S. Zeitlinger J. Levine M.S. Paused Pol II coordinates tissue morphogenesis in the Drosophila embryo.Cell. 2013; 153: 976-987Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar), and diseases ranging from cancer (Lin et al., 2010Lin C. Smith E.R. Takahashi H. Lai K.C. Martin-Brown S. Florens L. Washburn M.P. Conaway J.W. Conaway R.C. Shilatifard A. AFF4, a component of the ELL/P-TEFb elongation complex and a shared subunit of MLL chimeras, can link transcription elongation to leukemia.Mol. Cell. 2010; 37: 429-437Abstract Full Text Full Text PDF PubMed Scopus (426) Google Scholar, Miller et al., 2017Miller T.E. Liau B.B. Wallace L.C. Morton A.R. Xie Q. Dixit D. Factor D.C. Kim L.J.Y. Morrow J.J. Wu Q. et al.Transcription elongation factors represent in vivo cancer dependencies in glioblastoma.Nature. 2017; 547: 355-359Crossref PubMed Scopus (117) Google Scholar) to viral pathogenesis (Wei et al., 1998Wei P. Garber M.E. Fang S.M. Fischer W.H. Jones K.A. A novel CDK9-associated C-type cyclin interacts directly with HIV-1 Tat and mediates its high-affinity, loop-specific binding to TAR RNA.Cell. 1998; 92: 451-462Abstract Full Text Full Text PDF PubMed Scopus (1048) Google Scholar, Yamaguchi et al., 2001Yamaguchi Y. Filipovska J. Yano K. Furuya A. Inukai N. Narita T. Wada T. Sugimoto S. Konarska M.M. Handa H. Stimulation of RNA polymerase II elongation by hepatitis delta antigen.Science. 2001; 293: 124-127Crossref PubMed Scopus (128) Google Scholar). Precisely how RNAPII promoter-proximal pausing is enforced and regulated remains unclear; however, protein complexes, such as NELF and DSIF, increase pausing, whereas the activity of CDK9 (P-TEFb complex) correlates with pause release (Kwak and Lis, 2013Kwak H. Lis J.T. Control of transcriptional elongation.Annu. Rev. Genet. 2013; 47: 483-508Crossref PubMed Scopus (272) Google Scholar). Although much has been learned about RNAPII promoter-proximal pausing and its regulation, the underlying molecular mechanisms remain enigmatic. One reason for this is the complexity of the human RNAPII transcription machinery, which includes the ∼4.0 MDa PIC and many additional regulatory factors. Another underlying reason is that much current understanding derives from cell-based assays, which are indispensable but cannot reliably address mechanistic questions. For instance, factor knockdowns or knockouts cause unintended secondary effects and the factors and biochemicals present at each gene in a population of cells cannot possibly be defined. In vitro assays can overcome such limitations, but these have typically involved nuclear extracts, which contain a similarly undefined mix of proteins, nucleic acids, and biochemicals. To circumvent these issues, we sought to reconstitute RNAPII promoter-proximal pausing entirely from purified human factors (no extracts). Success with this task enabled us to address some basic mechanistic questions and opens the door for future studies to better define the contribution of specific factors in RNAPII promoter-proximal pause regulation. Past results in Drosophila and mammalian cells and extracts implicated the NELF, DSIF, and P-TEFb complexes as regulators of RNAPII pausing (Core et al., 2012Core L.J. Waterfall J.J. Gilchrist D.A. Fargo D.C. Kwak H. Adelman K. Lis J.T. Defining the status of RNA polymerase at promoters.Cell Rep. 2012; 2: 1025-1035Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar, Li et al., 2013Li J. Liu Y. Rhee H.S. Ghosh S.K. Bai L. Pugh B.F. Gilmour D.S. Kinetic competition between elongation rate and binding of NELF controls promoter-proximal pausing.Mol. Cell. 2013; 50: 711-722Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, Marshall and Price, 1992Marshall N.F. Price D.H. Control of formation of two distinct classes of RNA polymerase II elongation complexes.Mol. Cell. Biol. 1992; 12: 2078-2090Crossref PubMed Scopus (241) Google Scholar). We purified these factors in addition to the PIC factors TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH, Mediator, and RNAPII (Figure S1). Experiments were completed with the native human HSP70 promoter (HSPA1B gene), because others have shown that it is a quintessential model for promoter-proximal RNAPII pausing (Core et al., 2012Core L.J. Waterfall J.J. Gilchrist D.A. Fargo D.C. Kwak H. Adelman K. Lis J.T. Defining the status of RNA polymerase at promoters.Cell Rep. 2012; 2: 1025-1035Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). Because chromatin per se does not appear to be an essential regulator of RNAPII pausing in Drosophila or mammalian cells (Kwak et al., 2013Kwak H. Fuda N.J. Core L.J. Lis J.T. Precise maps of RNA polymerase reveal how promoters direct initiation and pausing.Science. 2013; 339: 950-953Crossref PubMed Scopus (452) Google Scholar, Lai and Pugh, 2017Lai W.K. Pugh B.F. Genome-wide uniformity of human ‘open’ pre-initiation complexes.Genome Res. 2017; 27: 15-26Crossref PubMed Scopus (16) Google Scholar, Li et al., 2013Li J. Liu Y. Rhee H.S. Ghosh S.K. Bai L. Pugh B.F. Gilmour D.S. Kinetic competition between elongation rate and binding of NELF controls promoter-proximal pausing.Mol. Cell. 2013; 50: 711-722Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar), the in vitro transcription assays were completed on naked DNA templates (also see below). Using purified PIC factors, primer extension assays established that transcription initiation occurred at the annotated HSPA1B start site in vitro (Figure S2A), as expected. An overview of the transcription assay is shown in Figure 1A, which was based in part upon in vitro pausing assays with nuclear extracts (Marshall and Price, 1992Marshall N.F. Price D.H. Control of formation of two distinct classes of RNA polymerase II elongation complexes.Mol. Cell. Biol. 1992; 12: 2078-2090Crossref PubMed Scopus (241) Google Scholar, Qiu and Gilmour, 2017Qiu Y. Gilmour D.S. Identification of regions in the Spt5 subunit of DRB sensitivity-inducing factor (DSIF) that are involved in promoter-proximal pausing.J. Biol. Chem. 2017; 292: 5555-5570Crossref PubMed Scopus (20) Google Scholar, Renner et al., 2001Renner D.B. Yamaguchi Y. Wada T. Handa H. Price D.H. A highly purified RNA polymerase II elongation control system.J. Biol. Chem. 2001; 276: 42601-42609Crossref PubMed Scopus (144) Google Scholar). Following PIC assembly, transcription was initiated by adding ATP, guanosine triphosphate (GTP), and uridine triphosphate (UTP) at physiologically relevant concentrations, with a low concentration of cytidine triphosphate (CTP), primarily 32P-CTP. After 1 min, reactions were chased with a physiologically relevant concentration of cold CTP and transcription was allowed to proceed for an additional 9 min. These “pulse-chase” assays allow better detection of short (potentially paused) transcripts, which otherwise would be drowned out by elongated transcripts that invariably possess more incorporated 32P-C bases. By directly labeling all transcripts with 32P-CTP, the method is highly sensitive and allowed detection of transcripts of varied lengths; furthermore, the 32P-CTP pulse-chase protocol ensured that 32P-labeled transcripts resulted almost exclusively from single-round transcription (see STAR Methods). Control experiments confirmed that transcripts detected were driven by the HSP70 promoter (e.g., not any contaminating nucleic acid) and that transcription was dependent on added PIC factors, as expected (Figure S2B). A variety of methods have established that RNAPII pauses after transcribing 20–80 bases in Drosophila and mammalian cells (Jonkers et al., 2014Jonkers I. Kwak H. Lis J.T. Genome-wide dynamics of Pol II elongation and its interplay with promoter proximal pausing, chromatin, and exons.eLife. 2014; 3: e02407Crossref PubMed Scopus (342) Google Scholar, Kwak et al., 2013Kwak H. Fuda N.J. Core L.J. Lis J.T. Precise maps of RNA polymerase reveal how promoters direct initiation and pausing.Science. 2013; 339: 950-953Crossref PubMed Scopus (452) Google Scholar, Lai and Pugh, 2017Lai W.K. Pugh B.F. Genome-wide uniformity of human ‘open’ pre-initiation complexes.Genome Res. 2017; 27: 15-26Crossref PubMed Scopus (16) Google Scholar, Lee et al., 2008Lee C. Li X. Hechmer A. Eisen M. Biggin M.D. Venters B.J. Jiang C. Li J. Pugh B.F. Gilmour D.S. NELF and GAGA factor are linked to promoter-proximal pausing at many genes in Drosophila.Mol. Cell. Biol. 2008; 28: 3290-3300Crossref PubMed Scopus (168) Google Scholar, Li et al., 2013Li J. Liu Y. Rhee H.S. Ghosh S.K. Bai L. Pugh B.F. Gilmour D.S. Kinetic competition between elongation rate and binding of NELF controls promoter-proximal pausing.Mol. Cell. 2013; 50: 711-722Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, Muse et al., 2007Muse G.W. Gilchrist D.A. Nechaev S. Shah R. Parker J.S. Grissom S.F. Zeitlinger J. Adelman K. RNA polymerase is poised for activation across the genome.Nat. Genet. 2007; 39: 1507-1511Crossref PubMed Scopus (569) Google Scholar, Nechaev et al., 2010Nechaev S. Fargo D.C. dos Santos G. Liu L. Gao Y. Adelman K. Global analysis of short RNAs reveals widespread promoter-proximal stalling and arrest of Pol II in Drosophila.Science. 2010; 327: 335-338Crossref PubMed Scopus (306) Google Scholar, Zeitlinger et al., 2007Zeitlinger J. Stark A. Kellis M. Hong J.W. Nechaev S. Adelman K. Levine M. Young R.A. RNA polymerase stalling at developmental control genes in the Drosophila melanogaster embryo.Nat. Genet. 2007; 39: 1512-1516Crossref PubMed Scopus (574) Google Scholar). The HSPA1B promoter sequence used in our assays extended 216 base pairs beyond the transcription start site (TSS); thus, elongated transcripts would migrate on a sequencing gel between 100 and 216 nt and paused transcripts would be observed between 20 and 80 nt. Prior to testing DSIF/NELF and P-TEFb, we completed experiments with the PIC alone. As expected, elongated transcripts were prevalent; however, we observed short transcripts, between 20 and 80 nt, consistent with promoter-proximal RNAPII pausing (Figure 1B, lane 1). Potentially, these short transcripts could reflect premature termination. However, time course experiments showed that the shorter transcripts build up and then release over time (Figure 1C). In fact, the increase in elongated products between 5 and 10 min was equal to the loss of pause signal between 5 and 10 min, suggesting a transient pause followed by release into elongation (see Discussion). Addition of NELF/DSIF to the reconstituted transcription system increased the levels of the short transcripts (20–80 nt) while decreasing the elongated products (Figure 1B, lane 2); these data were consistent with established roles for NELF/DSIF in RNAPII pausing (Kwak and Lis, 2013Kwak H. Lis J.T. Control of transcriptional elongation.Annu. Rev. Genet. 2013; 47: 483-508Crossref PubMed Scopus (272) Google Scholar) and further suggested that the short transcripts represented promoter-proximal paused products. Addition of P-TEFb to reactions containing NELF/DSIF largely reversed the promoter-proximal pausing induced by NELF/DSIF (Figure 1B, lane 3); thus, P-TEFb appeared to increase RNAPII pause release in vitro, also consistent with current models (Kwak and Lis, 2013Kwak H. Lis J.T. Control of transcriptional elongation.Annu. Rev. Genet. 2013; 47: 483-508Crossref PubMed Scopus (272) Google Scholar). A pause index (PI) was calculated and averaged across replicate experiments (n = 8; Figure 1D), which showed that NELF/DSIF increased PI, whereas P-TEFb decreased PI, as expected. Because we were able to recapitulate pause enhancement with NELF/DSIF and pause release with P-TEFb at the native human HSPA1B promoter, this in vitro system appeared to reliably reconstitute basic mechanistic aspects of RNAPII promoter-proximal pausing. Whereas many potential questions could be addressed with this system, we focused on the unexpected result that promoter-proximal pausing was recapitulated with the PIC alone. We next tested whether RNAPII pausing would be dependent on a specific PIC factor. Although some factors could not be reliably evaluated given their requirement for transcription in this assay, removal of TFIIA, TFIIH, HSF1, or Mediator still supported transcription in vitro, although at reduced levels. These experiments showed little change in PI, suggesting that these factors were not required for RNAPII pausing in this assay (Figure S2C). We also addressed a potential dependence on the large, multi-subunit TFIID complex. Whereas RNAPII transcription was not supported by removal of TFIID, TBP can substitute for TFIID in vitro, provided that the DNA templates are not assembled into chromatin (Näär et al., 1998Näär A.M. Beaurang P.A. Robinson K.M. Oliner J.D. Avizonis D. Scheek S. Zwicker J. Kadonaga J.T. Tjian R. Chromatin, TAFs, and a novel multiprotein coactivator are required for synergistic activation by Sp1 and SREBP-1a in vitro.Genes Dev. 1998; 12: 3020-3031Crossref PubMed Scopus (172) Google Scholar). Strikingly, we observed that, when PICs were assembled with TBP instead of TFIID, transcription still occurred but promoter-proximal RNAPII pausing was lost (Figure 2A). These data implicated TFIID as a key PIC factor that enabled RNAPII promoter-proximal pausing. To test further, we replaced endogenous purified human TFIID with a complete TFIID complex generated by recombinant expression (Figure 2B). As shown in Figure 2C, the recombinant human TFIID complex performed similarly to endogenous TFIID, confirming that TFIID was required for RNAPII promoter-proximal pausing in vitro. Having established a TFIID dependence for RNAPII pausing, we sought to determine whether this activity could be attributed to any specific TFIID subunits. Human TFIID is approximately 1.4 MDa in size and contains TBP plus 13 different TBP-associated factors (TAFs), which are present in one or two copies each. The structures of human TFIID bound to promoter DNA reveal that lobe C—containing TAF1, TAF2, and TAF7—binds downstream DNA (Louder et al., 2016Louder R.K. He Y. López-Blanco J.R. Fang J. Chacón P. Nogales E. Structure of promoter-bound TFIID and model of human pre-initiation complex assembly.Nature. 2016; 531: 604-609Crossref PubMed Scopus (141) Google Scholar, Patel et al., 2018Patel A.B. Louder R.K. Greber B.J. Grünberg S. Luo J. Fang J. Liu Y. Ranish J. Hahn S. Nogales E. Structure of human TFIID and mechanism of TBP loading onto promoter DNA.Science. 2018; 362: eaau8872Crossref PubMed Scopus (86) Google Scholar). In particular, TAF1/2 interact with the downstream promoter element (DPE) and the motif ten element (MTE). At the HSPA1B promoter, these elements reside at template position +18 to +33 relative to the TSS (Figure S1; Vo Ngoc et al., 2017Vo Ngoc L. Wang Y.L. Kassavetis G.A. Kadonaga J.T. The punctilious RNA polymerase II core promoter.Genes Dev. 2017; 31: 1289-1301Crossref PubMed Scopus (77) Google Scholar). Because the DPE and MTE encompass part of the RNAPII pause region, we hypothesized that lobe C subunits might be important in regulation of RNAPII promoter-proximal pausing. To test this hypothesis, we expressed and purified an “S-TAF” TFIID complex that contained only a subset of TAFs (Figure S2D). The S-TAF complex contains TBP as well as lobe C subunits TAF1 and TAF7. As shown in Figure S2D, the S-TAF complex was able to support pausing, implying a role for TFIID lobe C for this function. To further test the hypothesis that TFIID enables RNAPII promoter-proximal pausing, we turned to cell-based assays. To circumvent confounding issues with prolonged knockdown of essential TFIID subunits, we utilized the Trim-Away method (Clift et al., 2017Clift D. McEwan W.A. Labzin L.I. Konieczny V. Mogessie B. James L.C. Schuh M. A method for the acute and rapid degradation of endogenous proteins.Cell. 2017; 171: 1692-1706.e18Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar), which enabled rapid (t = 60 min) TAF subunit depletion (Figures 3A, 3B, and S3). (Numerous antibodies to various TAF subunits were tested prior to identification of a TAF1 antibody that reliably immunoprecipitated TFIID from extracts and depleted TAF1 using the Trim-Away protocol.) With this approach, the effect of TFIID could be evaluated with minimal compensatory or cytotoxic consequences. Indicative of a direct TAF1-TAF2 interaction in lobe C, Trim-Away experiments targeting TAF1 also depleted TAF2 (TAF7 was not probed due to lack of reliable antibodies), and other TFIID subunits were depleted to varying degrees, except for TBP (Figures 3B and S3). Because Trim-Away works through lysine modification by an E3 ubiquitin ligase, the enhanced TAF2 depletion versus TAF1 may result from its disordered C terminus, which contains 14 lysines in a 30-residue stretch. Following acute TAF depletion using Trim-Away, we isolated nuclei and performed replicate PRO-seq experiments (TAF1/2 knockdown versus controls). The data showed good correlation between replicates (Figures S4A and S4B), and normalization tests (see STAR Methods) confirmed that rapid TAF1/2 knockdown did not dramatically shift overall transcription levels versus controls (Figure 3C). An expectation based upon our in vitro results (Figures 2A and 2C) and cryoelectron microscopy (cryo-EM) structural data (Louder et al., 2016Louder R.K. He Y. López-Blanco J.R. Fang J. Chacón P. Nogales E. Structure of promoter-bound TFIID and model of human pre-initiation complex assembly.Nature. 2016; 531: 604-609Crossref PubMed Scopus (141) Google Scholar, Patel et al., 2018Patel A.B. Louder R.K. Greber B.J. Grünberg S. Luo J. Fang J. Liu Y. Ranish J. Hahn S. Nogales E. Structure of human TFIID and mechanism of TBP loading onto promoter DNA.Science. 2018; 362: eaau8872Crossref PubMed Scopus (86) Google Scholar) was that TFIID might serve as a “brake” for promoter-associated RNAPII complexes and that removal of this brake would enhance pause release. This expectation was confirmed by the PRO-seq data, which showed an overall increase in transcription genome-wide (Figures S4C–S4E), except at non-annotated enhancer RNAs (eRNAs) (see below). A representative protein-coding gene example is shown in Figure 3D, and genome-wide trends are shown as MA plots in Figures 3E and 3F. As might be expected from rapid depletion of TFIID lobe C subunits, the PRO-seq data showed transcriptional changes at thousands of gene 5′ ends. In cells, increased pause release can also promote re-initiation by additional RNAPII enzymes (Gressel et al., 2017Gressel S. Schwalb B. Decker T.M. Qin W. Leonhardt H. Eick D. Cramer P. CDK9-dependent RNA polymerase II pausing controls transcription initiation.eLife. 2017; 6: e29736Crossref PubMed Scopus (112) Google Scholar, Shao and Zeitlinger, 2017Shao W. Zeitlinger J. Paused RNA polymerase II inhibits new transcriptional initiation.Nat. Genet. 2017; 49: 1045-1051Crossref PubMed Scopus (122) Google Scholar). In agreement, we observed increased 5′ end reads at thousands of genes (Figure 3F), and the reads extended beyond promoter-proximal pause regions (Figure 4A, inset), suggesting a defect in pause enforcement. Unexpectedly, however, transcription sharply declined at approximately +300 downstream of the TSS, as shown at JUN and HSPA1B (Figures 3D and 4A, inset) and in a metagene plot representing all genes (Figures 4A and S5). We note that the sharp decline in reads beyond the promoter-proximal pause site superficially resembles RNAPII pausing; however, comparisons with PRO-seq data from flavopiridol-treated cells (a positive control for RNAPII pausing) showed stark differences and confirmed that pausing was not increased in TAF1/2-depleted cells (Figure S6). The sharp decline in transcription at around +300 in TAF1/2-depleted cells explains the reduced increase in gene body reads (Figure 3E) compared with 5′ end reads (Figure 3F) and suggests the presence of a distinct factor(s) that functions at this later, post-pause release stage (see Discussion). In contrast to annotated genes, transcription of non-annotated eRNAs declined overall in TAF1/2-depleted cells (Figure 4B), suggesting alternate regulatory mechanisms at these loci. As expected, a significant decrease in the TAF1 motif was observed in the Trim-Away depleted cells (Figure S6D). Taf1 knockdown in Drosophila S2 cells has minimal impact on other TFIID subunits (Pennington et al., 2013Pennington K.L. Marr S.K. Chirn G.W. Marr 2nd, M.T. Holo-TFIID controls the magnitude of a transcription burst and fine-tuning of transcription.Proc. Natl. Acad. Sci. USA. 2013; 110: 7678-7683Crossref PubMed Scopus (17) Google Scholar, Wright et al., 2006Wright K.J. Marr 2nd, M.T. Tjian R. TAF4 nucleates a core subcomplex of TFIID and mediates activated transcription from a TATA-less promoter.Proc. Natl. Acad. Sci. USA. 2006; 103: 12347-12352Crossref PubMed Scopus (117) Google Scholar), and promoter-proximal pausing is widespread in Drosophila (Muse et al., 2007Muse G.W. Gilchrist D.A. Nechaev S. Shah R. Parker J.S. Grissom S.F. Zeitlinger J. Adelman K. RNA polymerase is poised for activation across the genome.Nat. Genet. 2007; 39: 1507-1511Crossref PubMed Scopus (569) Google Scholar, Nechaev et al., 2010Nechaev S. Fargo D.C. dos Santos G. Liu L. Gao Y. Adelman K. Global analysis of short RNAs reveals widespread promoter-proximal stalling and arrest of Pol II in Drosophila.Science. 2010; 327: 335-338Crossref PubMed Scopus (306) Google Scholar, Zeitlinger et al., 2007Zeitlinger J. Stark A. Kellis M. Hong J.W. Nechaev S. Adelman K. Levine M. Young R.A. RNA polymerase stalling at developmental control genes in the Drosophila melanogaster embryo.Nat. Genet. 2007; 39: 1512-1516Crossref PubMed Scopus (574) Google Scholar). To further test the impact of TAF1 (a TFIID lobe C subunit) on RNAPII pausing, Taf1 was knocked down in S2 cells and PRO-seq experiments were completed in triplicate (Figures 4C and S7A). Consistent with TAF1/2-depleted human cells, Taf1 knockdown in Drosophila S2 cells showed a similar promoter-proximal increase in transcription genome-wide (Figure 4C), suggesting increased pause release and increased re-initiation with Taf1 knockdown (Figure S7B). These data suggest a conserved role for TFIID in the regulation of RNAPII promoter-proximal pausing. Structural data indicate that TFIID lobe C subunits TAF1 and TAF2 bind promoter DNA downstream of the TSS (Louder et al., 2016Louder R.K. He Y. López-Blanco J.R. Fang J. Chacón P. Nogales E. Structure of promoter-bound TFIID and model of human pre-initiation complex assembly.Nature. 2016; 531: 604-609Crossref PubMed Scopus (141) Google Scholar, Patel et al., 2018Patel A.B. Louder R.K. Greber B.J. Grünberg S. Luo J. Fang J. Liu Y. Ranish J. Hahn S. Nogales E. Structure of human TFIID and mechanism of TBP loading onto promoter DNA.Science. 2018; 362: eaau8872Crossref PubMed Scopus (86) Google Scholar). Past studies revealed that insertion of 10-bp DNA at the +15 site relative to the TSS disrupted RNAPII pausing at the HSP70 gene in Drosophila S2 cells (Kwak et al., 2013Kwak H. Fuda N.J. Core L.J. Lis J.T. Precise maps of RNA polymerase reveal how promoters direct initiation and pausing.Science. 2013; 339: 950-953Crossref PubMed Scopus (452) Google Scholar). This led to a “complex interaction” model for pausing, in which a promoter-bound factor(s) establishes an interaction (directly or indirectly) with the paused RNAPII complex. In agreement with this model, we observe a TFIID requirement for RNAPII promoter-proximal pausing in vitro, which is further supported by PRO-seq data in TAF-depleted human and Drosophila S2 cells. Additional evidence for TFIID-dependent regulation of RNAPII pausing derives from correlations among paused genes and DNA sequence elements bound by TFIID (Hendrix et al., 2008Hendrix D.A. Hong J.W. Zeitlinger J. Rokhsar D.S. Levine M.S. Promoter elements associated with RNA Pol II stalling in the Drosophila embryo.Proc. Natl. Acad. Sci. USA. 2008; 105: 7762-7767Crossref PubMed Scopus (126) Google Scholar, Lee et al., 2008Lee C. Li X. Hechmer A. Eisen M. Biggin M.D. Venters B.J. Jiang C. Li J. Pugh B.F. Gilmour D.S. NELF and GAGA factor are linked to promoter-proximal pausing at many genes in Drosophila.Mol. Cell. Biol. 2008; 28: 3290-3300Crossref PubMed Scopus (168) Google Scholar, Li et al., 2013Li J. Liu Y. Rhee H.S. Ghosh S.K. Bai L. Pugh B.F. Gilmour D.S. Kinetic competition between elongation rate and binding of NELF controls promoter-proximal pausing.Mol. Cell. 2013; 50: 711-722Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, Shao et al., 2019Shao W. Alcantara S.G. Zeitlinger J. Reporter-ChIP-nexus reveals strong contribution of the Drosophila initiator sequence to RNA polymerase pausing.eLife. 2019; 8: e41461Crossref PubMed Scopus (11) Google Scholar). Defects in TFIID function are linked to numerou" @default.
• W3011916202 created "2020-03-23" @default.
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• W3011916202 title "TFIID Enables RNA Polymerase II Promoter-Proximal Pausing" @default.
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• W3011916202 doi "https://doi.org/10.1016/j.molcel.2020.03.008" @default.
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